Chloride channels and transporters of the CLC family play crucial roles in a myriad of physiological processes including regulation of electrical excitability of nerve and muscle cells, modulation of salt and water movement across epithelia and acidification of intracellular compartments. The human genome encodes for 9 CLCs, 5 of which are transporters and 4 are channels. Mutations in 5 of these genes lead to the synthesis of proteins with altered functionalities that cause genetically inherited disorders such as myotonia congenita, Bartter's syndrome, Dent's disease, osteopetrosis and epilepsy. The involvement of these proteins in such a wide array of physiological and pathological processes marks them as ideal targets for the development of therapeutic treatments and drug design. This progress is, however, stunted by our lack of knowledge of the basic structural and mechanistic underpinnings underlying CLC function. This proposal aims to provide a molecular description of how CLC proteins regulate transmembrane Cl- fluxes and how these are coupled to H+ movement. This goal will be pursued through the combined use of X-ray crystallography, electrophysiological recordings, flux measurements and, for the first time for CLC proteins, direct substrate binding measurements. CLC proteins are homodimers where each monomer forms an independent permeation pathway. All family members catalyze movement of Cl- ions across cellular membranes, but can do so via either of two thermodynamically opposing mechanisms: the CLC channels dissipate the Cl- electrochemical gradient, whereas the CLC transporters catalyze uphill Cl- movement at the expense of the H+ electrochemical gradient, or vice versa. Our first major aim is to identify the molecular steps that allow CLC transporters to catalyze the stoichiometric exchange of Cl- and H+ across cellular membranes. Transporters undergo a complex series of conformational changes that allow them to transform the energy stored in electrochemical gradients into uphill substrate movement. We will identify, isolate and characterize the crucial structural players in this process. Our second major aim is to identify the molecular basis of anionic selectivity in CLC proteins. Substrate specificity is of paramount importance to proper function of both channels and transporters. We have now identified several residues potentially crucial for this process. We will test this hypothesis by manipulating through mutagenesis of these residues the selectivity of binding and permeability in order to alter the substrate specificity of the CLC channels and transporters. It has been proposed that CLC transporters and channels share a common architecture. The third goal of this proposal is to test this hypothesis by transforming the former into the latter. We will accomplish this by pursuing two complementary approaches: first, we will identify and eliminate the physical barriers blocking Cl- movement through the transporters and, second, we will identify the key residues differentiating the channels and the transporters and mutate the ones into the others. PUBLIC HEALTH RELEVANCE: The CLC channels and transporters mediate anion transport across cellular membranes to modulate the electrical excitability of muscle and nerve, to allow salt and water movement across epithelia, participate in acidification of vesicles along the endosomal- lysosomal pathway and of neurotransmitter release vesicles. Mutations in 5 of the 9 human CLC genes lead to genetic diseases. Because of the crucial role of these channels and transporters in all of these physiological processes, understanding the mechanism of function of these proteins will be therapeutically useful. By relating the structure of these proteins to their function, it may ultimately be possible to develop or identify pharmaceutical agents that could either enhance or inhibit Cl- transport, depending on the target and the ultimate goal.